Design, synthesis and biological evaluation of 1,4-Diazobicylco[3.2.2]nonane derivatives as α7-Nicotinic acetylcholine receptor PET/CT imaging agents and agonists for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.09.064

α7-Nicotinic acetylcholine receptor (α7-nAChR) agonists are promising therapeutic drug candidates for treating the cognitive impairment associated with Alzheimer's disease (AD). Thus, a novel class of derivatives of 1,4-diazobicylco[3.2.2]nonane has been synthesized and evaluated as α7-nAChR ligands. Five of them displayed high binding affinity (K_i = 0.001–25 nM). In particular, the K_i of 14 was 0.0069 nM, which is superior to that of the most potent ligand that was previously reported by an order of magnitude. Four of them had high selectivity for α7-nAChRs over α4β2-nAChRs and no significant hERG (human ether-a-go-go-related gene) inhibition. Their agonist activity was also discussed preliminarily. One of the compounds, 15 (K_i = 2.98 ± 1.41 nM), was further radiolabeled with ¹⁸F to afford [¹⁸F]15 for PET imaging, which exhibited high initial brain uptake (11.60 ± 0.14%ID/g at 15 min post injection), brain/blood value (9.57 at 30 min post injection), specific labeling of α7-nAChRs and fast clearance from the brain. Blocking studies demonstrated that [¹⁸F]15 was α7-nAChR selective. In addition, micro-PET/CT imaging in normal rats further indicated that [¹⁸F]15 had obvious accumulation in the brain. Therefore, [¹⁸F]15 was proved to be a potential PET radiotracer for α7-nAChR imaging.

Full text of DOI:10.1016/j.ejmech.2018.09.064

Accepted Manuscript
Design, synthesis and biological evaluation of 1,4-Diazobicylco[3.2.2]nonane
derivatives as α7-Nicotinic acetylcholine receptor PET/CT imaging agents and
agonists for Alzheimer's disease
Shuxia Wang, Yu Fan, Huan Wang, Hang Gao, Guohua Jiang, Jianping Liu, Qianqian
Xue, Yueheng Qi, Mengying Cao, Bingchao Qiang, Huabei Zhang
PII:
S0223-5234(18)30842-0
10.1016/j.ejmech.2018.09.064
EJMECH 10775
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 March 2018
Revised Date: 20 September 2018
Accepted Date: 25 September 2018
Please cite this article as: S. Wang, Y. Fan, H. Wang, H. Gao, G. Jiang, J. Liu, Q. Xue, Y. Qi, M. Cao,
B. Qiang, H. Zhang, Design, synthesis and biological evaluation of 1,4-Diazobicylco[3.2.2]nonane
derivatives as α7-Nicotinic acetylcholine receptor PET/CT imaging agents and agonists for
Alzheimer's disease, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.064.
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ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal home page: www.e lsevier.com/locate/ejmech
Design, Synthesis and Biological Evaluation of 1,4-Diazobicylco[3.2.2]nonane
Derivatives as α7-Nicotinic Acetylcholine Receptor PET/CT Imaging Agents and
Agonists for Alzheimer's Disease
⊥
⊥
Shuxia Wang , Yu Fang , Huan Wang, Hang Gao, Guohua Jiang, Jianping Liu, Qianqian Xue, Yueheng
Qi, Mengying Cao, Bingchao Qiang, Huabei Zhang*
Key Laboratory of Radiopharmaceuticals of Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
α7-Nicotinic acetylcholine receptor (α7-nAChR) agonists are promising therapeutic drug candidates for
treating the cognitive impairment associated with Alzheimer's disease (AD). Thus, a novel class of
derivatives of 1,4-diazobicylco[3.2.2]nonane has been synthesized and evaluated as α7-nAChR ligands.
i i
Five of them displayed high binding affinity (K = 0.001-25 nM). In particular, the K of 14 was 0.0069
Available online
nM, which is superior to that of the most potent ligand that was previously reported by an order of
magnitude. Four of them had high selectivity for α7-nAChRs over α4β2-nAChRs and no significant
hERG (human ether-a-go-go-related gene) inhibition. Their agonist activity was also discussed
18
i
preliminarily. One of the compounds, 15 (K = 2.98 ± 1.41 nM), was further radiolabeled with F to
Keywords:
18
afford [ F]15 for PET imaging, which exhibited high initial brain uptake (11.60 ± 0.14%ID/g at 15 min
post injection), brain/blood value (9.57 at 30 min post injection), specific labeling of α7-nAChRs and fast
Alzheimer's Disease (AD)
α7-Nicotinic acetylcholine receptor (α7-
nAChR)
1
8
clearance from the brain. Blocking studies demonstrated that [ F]15 was α7-nAChR selective. In
1
8
addition, micro-PET/CT imaging in normal rats further indicated that [ F]15 had obvious accumulation
1
,4-Diazobicylco[3.2.2]nonane
1
8
in the brain. Therefore, [ F]15 was proved to be a potential PET radiotracer for α7-nAChR imaging.
2018 Elsevier Masson SAS. All rights reserved
Radiotracer
PET/CT imaging
©
.
1
. Introduction
antagonists α-bungarotoxin and methyllycaconitine (MLA) [7,
].
There is powerful evidence that α7-nAChRs play a critical role
8
Alzheimer's disease (AD), the most common form of dementia,
is widespread in people over 65 [1]. AD is an irreversible and
progressive neurodegenerative disease characterized by memory
impairment, cognitive dysfunction, language deterioration, etc.
in the pathogenesis of AD [9]. Many pharmacological and
genetic studies and clinical trials have shown the involvement of
α7-nAChRs in cognitive deficits. In human studies, reduction in
α7-nAChR binding sites and protein levels is observed in the
brains of patients with AD[10, 11]. Based on this research, there
is a strong possibility that α7-nAChR agonists are a potential
avenue to effectively improve cognitive function in patients with
AD [12].
[
2]AD also imposes a huge economic burden and induces
psychological pressure that is predicted to be unbearable.
However, the medicines currently available can provide only
modest and temporary improvement in cognitive performance
and are ineffective in curing AD.
Nicotinic acetylcholine receptors (nAChRs), a superfamily of
ligand-gated ion channels widely expressed in the central and
peripheral nervous systems [3], are pentameric structures
composed of α (i.e., α2−α10) and β (i.e., β2−β4) subunits that
assemble into various combinations as homopentamers or
heteropentamers [4] in response to the binding of the
neurotransmitter acetylcholine (ACh). Among the many subtypes
in the human brain, heteromeric α4β2-nAChRs and homomeric
α7-nAChRs are the most abundant [5]. α7-nAChRs are composed
of five identical α7 subunits and mainly exist in the
hippocampus, cerebral cortex and other regions associated with
learning, memory and cognition [6]. α7-nAChRs are also
For these reasons, α7-nAChRs as a significant drug target to
treat AD have received increasing interest in the fields of science
and medicine. Thus, great progress in quantity and quality of α7-
nAChR ligands has been made over the past decades (for reviews
[
2, 13, 14]), including GTS-21 [15], AR-R17779 [16],
SSR180711 [17], TC-5619 [18], BMS-902483 [19] (figure 1).
Although some of the ligands with better properties have been
proved to improve cognitive performance in animal models and
clinical trials, none have been put on the market because of their
limitation in structural diversity, affinity, selectivity or other side
effects.
Imaging and quantifying α7-nAChRs with positron-emission
tomography (PET) and single-photon emission computed
tomography (SPECT) might offer a non-invasive way to
2+
characterized by high permeability to Ca and affinity for the
�
Corresponding author.
E-mail address: hbzhang@bnu.edu.cn (Huabei Zhang)

critically facilitate the early diagnosis of α7-nAChR-related CNS
although the test conditions were not exactly the same. The
affinities of the two fluoro ligands, 6 and 15, were also high and
were comparable to that of MLA. Fluoro derivative 24 also
showed moderate binding affinity whereas the affinity of isomer
25 was much lower.
ACCEPTED MANUSCRIPT
disorders and provide an insight into its role in AD and
schizophrenia. Many leading compounds have been labeled with
1
25
11
18
125
11
I, C or F [20, 21], such as [ I]1 [22], [ C]CHIBA-1001
18
18
[
23], [ F]NS10743 [24], and [ F]ASEM [25] (figure 1), but
most of them displayed low brain uptake or specific binding. The
18
most recent α7-nAChR PET tracer, [ F]ASEM with high
specific affinity for α7-nAChR (K = 0.4 nM), which was
i
developed by Gao et al. [25], has been carried out in human
18
studies [26, 27], and homologous [ F]DBT10 [27] (K = 1.3 nM)
i
shows similar characteristic. These two compounds are the most
promising agents for human α7-nAChR PET imaging.
1
,4-Diazobicylco[3.2.2]nonane as a good skeleton are widely
found in α7-nAChR ligands [28, 29]. And on the basis of our
previous work [30, 31], herein, we report the design and
synthesis of a new derivatives of 1,4-diazobicylco[3.2.2]nonane
and their evaluation as α7-nAChR ligands based on their affinity,
agonist activity, hERG toxicity and selectivity. Moreover,
radiochemical techniques were used to label one of these
18
18
compounds, 15 [32], with [ F] to afford [ F]15 which was
biologically evaluated in mice for PET imaging of α7-nAChRs.
2
. Results and Discussion
.1. Chemistry
The synthesis of the meta-substitution compounds 5, 6 is
Figure 1. Representative α7-nAChRs ligands and radioligands.
2
Heteromeric α4β2-nAChRs is the main cerebral subtype, so
the binding assay for α4β2-nAChRs was also performed using
3
outlined in scheme 1. Bromination and nitration of the starting
material 9H-fluoren-9-one (1) provided 2-bromo-7-nitro-9H-
fluoren-9-one (3), which was sequentially reacted with 1,4-
diazabicyclo[3.2.2]nonane via the Buchwald−Hartwig cross-
coupling reaction to afford 2-(1,4-diaza-bicyclo[3.2.2]nonan-4-
yl)-7-nitro-9H-fluoren-9-one (4). Then, reduction of the nitro
group in 4 with iron powder yielded the corresponding aniline 5.
[
H]cytisine (1nM) as the competing radioligand (table 1). 5 and 14
showed high selectivity because of strong affinity for α7-nAChRs.
The fluoro derivatives 6 and 15 were also displayed good α7-
/α4β2-nAChRs selectivity. Because of the excellent affinity and
selectivity of these four ligands, their agonistic activities were
preliminarily elucidated by a patch clamp electrophysiology
assay. The result showed that none of them were strong agonists
to acetylcholine ligand-gated channels at 1 µM relative to
acetylcholine at 30 µM (the data analysis is shown in the
supporting information).
6
was obtained via the Schiemann reaction.
The synthesis of the ortho-substitution compounds is outlined
in scheme 2. Fluoranthene (7) was oxidized by chromium (VI)
oxide and brominated to 7-bromo-9-oxo-9H-fluorene-1-
carboxylic acid (9) in high yields. The carboxy group in 9 was
Table 1
Binding affinities (K
i
,
nM) of MLA, nicotine and the 1,4-
converted to
a Boc-protected aniline using the Curtius
diazobicylco[3.2.2]nonane derivatives for α7-nAChRs and α4β2-nAChRs
rearrangement followed by tert-butyl ester formation in one pot
to afford tert-butyl 2-bromo-9-oxo-9H-fluoren-8-ylcarbamate
K
i
(nM)
Selectivity
α7/α4β2
Comp.
a
b
α7
α4β2
nt
(10). In addition, 10 was treated with 1 M HCl to remove the N-
c
MLA
Nicotine
2.88 ± 0.78
Boc-protecting group and oxidized with 30% H O providing 7-
bromo-1-nitro-9H-fluoren-9-one (12). The desired compounds
1
6
c
2
2
nt
4.1 ± 1.13
4652 ± 798
4734 ± 653
6656 ± 812
3693 ± 325
5
6
0.064 ± 0.058
7.24 ± 1.02
0.0069 ± 0.004
2.98 ± 1.41
21.76 ± 1.22
377.94 ± 118.534
>10000
654
>10000
1239
4 and 15 were ultimately prepared using similar steps as 5 and
.
The synthesis of compounds 24, 25 is outlined in scheme 3.
14
5
4
1
2
2
c
nt
nt
The compound 21 was obtained through the methods previously
reported [28].
c
5
a
125
SD rat (female) cerebral cortex; radiotracer, [ I]α-bungarotoxin (0.4 nM),
K
D
= 0.7 nM; K
i
values are the means ± SD of three experiments performed
2
.2. In vitro binding assay and agonistic effects
in triplicate.
b
3
SD rat (female) cerebral cortex; radiotracer, [ H]Cytisine (1 nM), K
D
= 0.77
To determine the affinity of the synthesized compounds for
α7-nAChRs, in vitro competition binding assays were performed
with extracted rat cerebral cortex as the receptor protein and
nM; K
i
values are the means ± SD of three experiments performed in
triplicate.
c
nt: not determined.
1
25
[
I]α-bungarotoxin (0.4 nM), an α7-nAChR antagonist (KD
=
2
.3. hERG assay
0
.7 nM), as the competing radioligand. Furthermore, MLA, an
α7-nAChR-selective antagonist, was also tested for confirming
the reliability of the method (table 1) and for comparison. Five
compounds exhibited remarkable binding affinities for α7-
Inhibition of the hERG potassium channels may introduce QT
interval prolongation, thereby causing serious cardiac side effects.
Some famous drugs, such as terfenadine, astemizole,
grepafloxacin and cisapride, were withdrawn from the market
due to their hERG inhibitory activity [33]. Recognizing hERG
inhibition in an early phase is an indispensable component in
drug discovery. We determined the binding affinity of the
reference molecule cisapride and the four ligands for hERG
channels by a traditional patch clamp technique (table 2). Two
nAChRs (K = 0.001-25 nM). The two amino derivatives, 5 and
i
1
4, showed two and three orders of magnitude better affinities
than the reference ligand MLA, respectively. In particular, 14 (Ki
=
0.0069 ± 0.004 nM) still displayed exceptional binding to the
target: its affinity was superior to that of the most potent ligand by
an order of magnitude, as indicated by a rough comparison with
the highest affinity compound (K = 0.023 nM [29], 0.3 nM [25]),
i
2

amino derivatives 5 and 14 had no significant affinity for hERG
potassium channels with less than 10000 selectivity over
ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of meta-substitution compounds. (a) Br
2
, 10% NaHSO3, 80-85°C; (b) HNO , H
3
2 4 2 3
SO ; (c) Pd (dba) , BINAP, 1,4-diazabicyclo[3.2.2]nonane,
t-BuONa, 1,4-dioxane, 80-85°C; (d) Fe, CH CH OH, conc. HCl, 80°C; (e) HBF
3
2
4
, 0-5°C; NaNO ; xylene, 120°C.
2
Scheme 2. Synthesis of ortho-substitution compounds. (a) CrO
BuOH, 110°C; (d) CH CN, 1 M HCl, 82°C, 1 M NaOH; (e) 30% H
Cs CO , toluene, 80-85°C; (g) Fe, CH CH OH, conc. HCl, 80°C; (h) HBF
3
, H
2
O, CH
, (CF
, 0-5°C; NaNO
2
3
COOH, 80-85°C, 110-120°C; (b) Br
CO) O, CH Cl , 0°C-RT; (f) Pd
; xylene, 120°C.
2
, 80-85°C; (c) toluene, (CH
3 2 3
CH ) N, DPPA, t-
3
2
O
2
3
2
2
2
2
(dba) , BINAP, 1,4-diazabicyclo[3.2.2]nonane,
3
2
3
3
2
4
Scheme 3. (a) CH
CH CH N, (CH
3
ONa, RT; Br
2
, 0°C; (b) Zn, NH
4
Cl, 50°C; (c) CS
2 2 3 3
, KOH, 80-85°C; (d) K CO , DMF, 0°C, CH I; (e) 1,4-diazabicyclo[3.2.2]nonane,
(
3
2
)
3
3
)
2
CHOH, 100°C; (f) Cs
2
CO , Pd(dppf)Cl
3
2
, 1,4-dioxane, 85-90°C.
α7-nAChRs. 15 showed modest cross-activity with hERG
channels but good separation from its affinity for α7-nAChRs.
The compound 6, however, was found to have strong hERG
inhibition because of its relative low affinity for α7-nAChRs.
In all fluoro derivatives we synthesized, 15 had the best
binding affinity and high selectivity for α7-nAChRs.
18
Furthermore, the radiolabeling of [ F]15 can be accomplished
18
by a direct nucleophilic substitution (S Ar) with [ F]fluoride via
N
the nitro 13 because the leaving nitro group in 13 is activated for
SNAr fluorination by the electron-withdrawing COAr on the
ortho position [25, 34]. Therefore, fluoro derivative 15 was
selected for labeling and further evaluation as a PET radiotracer
for α7-nAChR imaging.
Table 2
hERG inhibition (IC50, nM) of cisapride and the ligands
hERG
IC50(nM)
α7-nAChRs
IC50(hERG)/
Compd
a
b
IC50(nM)
IC50(α7-nAChRs)
c
c
Cisapride 13.8 ± 0.76
nt
nt
5
6
1
1
2500 ± 150
460 ± 55
49700 ± 15900
460 ± 11
0.21 ± 0.19
20.86 ± 2.94
0.023 ± 0.013
6.23 ± 2.94
>10000
22
>10000
74
2.4. Acute toxicity studies
4
5
To evaluate the safety profile of 15, Kunming mice (half male
and half female, 18-22 g, n = 60) were administered different
concentrations of 15 (0.1 mL) via the tail vein, whereas the
a
b
c
IC50 values are the means ± SD of three experiments.
from an in vitro binding assay.
nt: not determined.
3

control group was injected only with the vehicle solution. The
mice were monitored and recorded over 14 days. There was no
ACCEPTED MANUSCRIPT
A
B
1
8
18
Figure 2. Radiosynthesis. (A) The radiosynthetic route of [ F]15. (B) HPLC chromatogram of co-injection of [ F]15 and 15.
18
results are summarized in table 3. [ F]15 exhibited high initial
brain uptake with 8.98 ± 0.41 %ID/g at 5 min post injection, and
morbidity or mortality in control group. In the group treated with
5, some mice showed different degrees of convulsions,
1
1
1.60 ± 0.14 %ID/g, its highest uptake, at 15 min post injection.
vomiting blood or death. The LD50 value of 15 was 53.70 mg/kg,
as calculated by the weighted probit analysis developed by Bliss,
which is far greater than a PET imaging dose (µg/human),
The brain15min/brain90min value was 3.20, a reasonable clearance
rate for brain PET imaging. Among the peripheral organs, the
blood uptake was low, resulting in a high brain/blood ratio of
18
indicating that the homologous radioligand [ F]15 is safe for use
9
.57 at 30 min post injection. The lung displayed supernormal
in PET imaging and is worthy of further study.
initial uptake with 70.52 ± 7.86 %ID/g at 5 min post injection
but fast clearance with 20.23 ± 1.51 %ID/g at 15 min post
injection and 4.99 ± 0.45 %ID/g at 90 min post injection.
Notably, the bone uptake increased from 3.02 ± 0.11 %ID/g to
2
.5. Radiochemistry
Compound 15 with high affinity and selectivity was chosen for
18 -
radiolabeling with F in one step by treating nitro precursor 13
7
.65 ± 0.52 %ID/g within 90 min post injection demonstrating
18 -
18
with F /K C O and Kryptofix 222 in anhydrous DMSO at
2
2
4
the slight in vivo defluorination of [ F]15
.
1
60°C for 30 min (figure 2) and was purified by high-
performance liquid chromatography (HPLC) in 13.1%
radiochemical yield (decay not corrected) with a radiochemical
purity greater than 98%. The total synthesis time was
approximately 60 min. [ F]15 was identified by comparison of
its retention time with that of nonradioactive 15 via co-injection.
2.8. Biodistribution in brain regions of mice
18
The distribution of [ F]15 in the brain of normal Kunming
mice (28-32 g, female) was studied (figure 4). The highest
accumulation of radioactivity was observed in the frontal cortex
18
(
9.39 ± 0.24 %ID/g), striatum (8.37 ± 0.27 %ID/g) and
2
.6. Octanol−water partition coefficient and in vitro stability
hippocampus (6.31 ± 0.82 %ID/g), with peak uptake at 30 min
after injection, followed by a decrease. The lowest uptake was
found in the cerebellum, and the radioactivity in the
superior/inferior colliculus and thalamus was moderate. The
18
The ability of [ F]15 to permeate the blood−brain barrier
BBB) was determined by estimating the partition coefficient
logP) in an n-octanol/phosphate buffered saline (PBS, pH = 7.4)
system. The logP of [ F]15 was 1.64 ± 0.12, which falls in a
desirable range (logP = 1-3) and thus indicates increased brain
uptake and decreased nonspecific binding [35, 36].
The in vitro stability of [ F]15 in fetal bovine serum and
saline was tested by measuring the radiochemistry purity via
HPLC after incubation at 37°C and r.t., respectively. The
radiochemistry purity in fetal bovine serum and saline after 1 h
and 2 h was all greater than 98% (figure 3), indicating the high in
(
(
18
distribution of [ F]15 in the brain was consistent with the
distribution of α7-nAChRs in the brain reported in the literature
18
18
[
20]. Moreover, the clearance rate of [ F]15 from the cerebellum
was higher than that from any other regions. Compared to the
18
18
18
18
most promising ligands [ F]ASEM and F-DBT10, [ F]15
showed similar distribution and higher peak uptake in the α7-
nAChR-rich regions. The highest regional BPND value (Table 4)
at 90 min (2.6 in hippocampus) was lower than that of
1
8
[
F]ASEM (8.0 in the superior and inferior colliculus) and
18
18
vitro biostability of [ F]15
.
comparable to that of [ F]DBT10 (3.1 in the hippocampus),
probably because the binding affinity of [ F]15 (K = 2.98 nM) is
weaker than that of [ F]ASEM (K = 0.4 nM) and similar to that
18
i
18
2
.7. Biodistribution in tissues and organs of mice
i
18
18
of [ F]DBT10 (K = 1.3 nM); nevertheless, the highest regional
i
18
The biodistribution of [ F]15 after intravenous injection was
evaluated in normal Kunming mice (18-22 g, female), and the
BPND value of [ F]15 is still better than that of other ligands,
which in general are < 2.0.
4

ACCEPTED MANUSCRIPT
18
Figure 3. HPLC chromatograms of [ F]15 in fetal bovine serum and saline. (A) Fetal bovine serum, 37°C, 1 h. (B) Fetal bovine serum, 37°C, 2 h. (C) Saline, RT,
h. (D) Saline, RT, 2 h.
1
Table 3
18
a
Biodistribution of [ F]15 in Kunming mice (18-22 g, female)
time (min)
30 min
organs
5
min
15 min
60 min
90 min
blood
brain
heart
liver
spleen
lung
kidney
muscle
bone
1.43 ± 0.06
8.98 ± 0.41
8.37 ± 0.14
8.19 ± 1.19
7.72 ± 0.65
70.52 ± 7.86
15.92 ± 0.24
5.51 ± 0.65
3.02 ± 0.11
6.23
1.24 ± 0.16
11.60 ± 0.14
4.45 ± 0.62
12.28 ± 1.96
11.58 ± 0.92
20.23 ± 1.51
11.54 ± 0.79
3.48 ± 0.40
4.55 ± 0.68
9.35
1.03 ± 0.07
9.86 ± 0.05
3.56 ± 0.31
13.74 ± 1.36
8.67 ± 0.48
18.30 ± 1.56
8.06 ± 0.69
2.36 ± 0.30
4.84 ± 0.18
9.57
0.68 ± 0.01
5.46 ± 0.27
2.19 ± 0.16
9.74 ± 0.07
4.08 ± 0.38
7.64 ± 0.99
4.85 ± 0.443
1.79 ± 0.06
7.27 ± 1.31
8.03
0.52 ± 0.06
3.63 ± 0.25
1.71 ± 0.13
7.46 ± 0.19
2.97 ± 0.25
4.99 ± 0.45
4.26± 0.19
1.32 ± 0.11
7.65 ± 0.52
6.98
brain/blood
a
Data are expressed as the percentage of injected dose per gram (%ID/g), mean ± SD, n = 5.
1
8
Figure 4. Biodistribution of [ F]15 in the brain regions of Kunming mice (28-32 g, female). Data are expressed as the percentage of injected dose per gram
%ID/g), mean ± SD, n = 5. Abbreviations: Coll, superior and inferior colliculus; Hipp, hippocampus; FrCtx, frontal cortex; Rest, rest of the brain; Th, thalamus;
Str, striatum; CB, cerebellum.
(
5

ACCEPTED MANUSCRIPT
Table 4
a
18
The BPND values of different tissues in Kunming mice (28-32 g, female) of [ F]15
BPND
tissue
5
0.24
nt
0.2
nt
nt
min
15 min
0.63
0.20
0.28
0.28
0.11
30 min
1.07
0.39
0.85
0.31
0.28
60 min
1.29
1.36
0.87
0.45
0.82
90 min
1.47
2.63
1.86
0.86
1.10
FrCtx
Hipp
Str
Coll
Th
b
b
b
a
BPND = (regional uptake/cerebellum uptake) -1[37].
nt: not determined.
b
Abbreviations: Coll, superior and inferior colliculus; Hipp, hippocampus; FrCtx, frontal cortex; Rest, rest of brain; Th, thalamus; Str, striatum; CB, cerebellum.
18
2
.9. Blocking studies in CD-1 mice
binding of [ F]15 to α7-nAChRs was selective over its binding
to α4β2-nAChRs and 5-HT3.
1
8
To further determine the specific binding of [ F]15 to α7-
nAChRs over other similar receptors, blocking studies were
carried out by pre-subcutaneous injection of blocking agents.
α4β2-nAChRs, a heteromeric nAChR subtype widely expressed
in the brain [38], and 5-HT a receptor sharing high sequence
2
.10. Micro-PET/CT imaging studies in rats
18
[ F]15 was evaluated in small animal PET/CT imaging
studies. CD-1 rats (180-200 g, female) were injected
3,
18
homology with α7-nAChRs [39], were the most likely receptors
to cross-binding with α7-nAChR ligands. Therefore, cytisine (1
mg/kg, a selective partial agonist of α4β2-nAChRs) and
ondansetron (2 mg/kg, a selective antagonist of 5-HT ) were pre-
injected 5 min and 10 min prior to the intravenous injection of
intravenously with [ F]15 (0.3 mL, 200 µCi) and anesthetized
with isoflurane for imaging. Axial, coronal and sagittal PET
18
images of [ F]15 in a rat at 15, 30 and 60 min post injection are
shown in figure 6. In agreement with the biodistribution results
mentioned above, the highest tracer uptake in the brain was
observed at 15 min post-injection, and relatively high
accumulation remained at 30 min post injection. At 60 min after
injection, the tracer uptake in the brain was lower but was still
3
18
[
F]15, respectively. The mice were sacrificed at 60 min after
18
the [ F]15 injection, and the brain tissues were harvested. As
expected (figure 5), the accumulation of radiotracer in different
brain regions of the blocked-group showed almost no changes
compared to that in the control group, suggesting that the
1
8
noticeable. The imaging results further demonstrated that [ F]15
could be a potential PET radiotracer for in vivo imaging of α7-
nAChRs.
1
8
Figure 5. Blocking studies of [ F]15 accumulation in Kunming mouse brains (28-32 g, female) by pre-subcutaneous injection of cytisine (1 mg/kg) and
ondansetron (2 mg/kg). Data are expressed as the percentage of injected dose per gram (%ID/g), mean ± SD, n = 5. Abbreviations: Coll, superior and inferior
colliculus; Hipp, hippocampus; FrCtx, frontal cortex; Rest, rest of brain; Th, thalamus; Str, striatum; CB, cerebellum.
6

ACCEPTED MANUSCRIPT
18
Figure 6. Micro-PET/CT images of [ F]15 in CD-1 (180-200 g, female) rats.
3
. Conclusion
quadrupole mass spectrometer. HPLC analysis and purification
®
were performed on a Shimadzu LC-20AT system equipped with
A series of 1,4-diazobicylco[3.2.2]nonane derivatives was
®
an SPD-20A UV detector (λ = 280 nm) and a Bioscan flow
prepared and evaluated as α7-nAChR-targeted ligands. Five of
them had high binding affinity for α7-nAChR (K = 0.001-25
nM), especially the compound 14 (K = 0.0069 nM). And four of
them showed high selectivity for α7-nAChRs over α4β2-nAChRs. In
the preliminary study of agonistic activities, no strong agonist
activity was found at 1 µM relative to acetylcholine at 30 µM. 5
and 14 exhibited minimal binding affinity for the hERG
potassium channels, whereas 6 and 15 displayed slight cross-
binding with these channels but good separation from their
affinity for α7-nAChRs. In acute toxicity studies, the LD of 15
count 3200 NaI/PMT γ-radiation scintillation detector. HPLC
®
i
separations were achieved on an Inertsil ODS-3 C18 reverse
i
phrase semi-preparative column (GL Sciences, Inc. 5 µm, 10
mm×250 mm) with elution using a binary gradient system at a
flow rate of 4 mL/min. HPLC analysis was performed on a
Venusil XBP C18(L) reverse phrase analytical column (Agela
Technologies, 5 µm, 150 Å, 4.6×250 mm) with elution using a
binary gradient system at a flow rate of 1 mL/min. The purity of
all the synthesized compounds used for biological evaluation
were detected to be > 95% by an HPLC method. Radioactivity
50
(
(
53.70 mg/kg) was far greater than a PET imaging dose
µg/human).
2
was measured on a WIZARD 2480 automatic γ-counter
(
PerkinElmer, USA). SD rats (female, 180 - 200 g), normal
Kunming mice (18-22 g, 28-32 g, female) and CD-1 rats (female,
80-200 g) were provided by Beijing Xinglong Animal
Fluoro derivative 15 with good binding affinity (K = 2.98 ±
.41 nM) was further radiolabeled with F to afford [ F]15 for
i
18
18
1
1
18
PET/CT imaging. [ F]15 displayed high in vitro stability and can
sufficiently permeate the BBB to specially label the α7-nAChRs
in the brain. Furthermore, micro-PET/CT imaging in normal rats
Technology Co, Ltd., and SD mice (female, 180-200 g) were
purchased from Beijing Vital River Animal Technology Co, Ltd.
All mice protocols were approved by the Animal Care
Committee of Beijing Normal University.
18
further indicated that [ F]15 had obvious accumulation in the
1
8
brain. Therefore, [ F]15 has potential to be a suitable α7-nAChR-
targeted imaging radiotracer for early AD diagnosis, and further
studies on this radioligand are ongoing.
4
4
.2. Chemistry
.2.1. 2-Bromo-9H-fluoren-9-one (2) [40]
4
. Experiments
Br (32.0 g, 0.2 mol) was added to a solution of 9H-fluoren-9-
2
one (1) (30.0 g, 0.17 mol) in 100 mL of H O dropwise at 80-
2
4
.1. Materials and measurements
8
5°C over 30 min and stirred for 4 h. After complete conversion
was detected by TLC, the reaction mixture was cooled to RT,
General procedures. All chemicals used in this work were
quenched by the addition of 10% NaHSO and then stirred for 30
3
commercial products and were utilized without further
min. The solid was filtered and dried to afford the corresponding
18 -
purification unless otherwise specified. F was obtained from
Peking Cancer Hospital (Beijing, China). Chemical reactions
were monitored by thin-layer chromatography (TLC) using silica
gel 60 GF254 and were visualized by a UV lamp at 254 nm. Flash
column chromatography was conducted on silica gel (Bonna-
compound 2-bromo-9H-fluoren-9-one (2) (39.1 g, 90.4%) as a
1
yellow solid. H NMR (400 MHz, CDCl ) δ 7.76(d, J = 1.64 Hz,
3
1
1
1
H), 7.66 (d, J = 7.36 Hz, 1H), 7.61 (dd, J = 7.84 Hz, 1.76 Hz,
H), 7.51-7.50 (m, 2H), 7.39 (d, J = 7.92 Hz, 1H), 7.35-7.29 (m,
H); MS (M+H ): m/z = 260.98.
+
®
Agela flash silica, 40-60 µm, 60 Å) using an Interchim
®
Puriflash 4100 medium pressure preparative chromatography
4
.2.2. 2-Bromo-7-nitro-9H-fluoren-9-one (3) [40]
®
apparatus. NMR spectra were recorded on a Bruker Avance III
A suspension of 2-bromo-9H-fluoren-9-one (2) (0.09 mol) in
HD 400 with chemical shifts (δ) expressed in parts per million
H O (180 mL) was heated to 80-85°C and a solution of 65%
2
(
ppm) relative to TMS as an internal standard at RT. Coupling
constants (J) are reported in hertz. Multiplicity is denoted by s
singlet), d (doublet), t (triplet), quartet (q), and m (multiplet)®.
MS spectra were recorded on a Waters Quattro Micro
HNO (2.6 mol, 180 mL) and 98% H SO (3.3 mol, 180 mL) was
3
2
4
added dropwise. The reaction mixture was heated to reflux and
(
continued for 4 h. After cooling to room temperature, H O (300
2
mL) was added and the mixture was filtered. The yellow cake
7

was washed with water (3×100 mL) and methanol (200 mL). The
7.24-7.19 (m, 3H), 7.05 (d, J = 2.28 Hz, 1H), 7.02 (dd, J = 8.4
Hz, 2.4 Hz, 1H), 6.74 (dd, J = 8.32 Hz, 2.56 Hz, 1H), 4.0437-
4.0391 (m, 1H), 3.55-3.52 (m, 2H), 3.15-3.07 (m, 4H), 3.01-2.94
ACCEPTED MANUSCRIPT
obtained solid was dried to provide the title product 2-bromo-7-
1
nitro-9H-fluoren-9-one (3) (21.0 g, 76.4%) as a yellow solid. H
13
NMR (400 MHz, CDCl ) δ 8.49 (s, 1H), 8.44 (d, J = 8.16 Hz,
(m, 2H), 2.12-2.05 (m, 2H), 1.77-1.68 (m, 2H); C NMR (101
3
1
1
H), 7.90 (s, 1H), 7.75 (d, J = 7.92 Hz, 1H), 7.71 (d, J = 8.16 Hz,
H), 7.55 (d, J = 7.92 Hz, 1H); MS (M+H ): m/z = 306.02.
MHz, CDCl ) δ 192.51 (d, J = 2.2 Hz), 162.49 (s), 160.03 (s),
148.88 (s), 140.55 (d, J = 2.9 Hz), 130.56 (s), 120.13 (s), 119.86
3
+
(s), 119.63 (s), 118.92 (s), 118.85 (s), 116.74 (s), 110.86 (s),
4
9
.2.3. 2-(1,4-Diazabicyclo[3.2.2]nonan-4-yl)-7-nitro-9H-fluoren-
-one (4) [41]
110.63 (s), 108.67 (s), 55.97 (s), 50.73 (s), 45.48 (s), 43.49 (s),
19
25.76 (s); F NMR (376 MHz, CDCl ) δ -115.21 (s); MS
3
+
2
-bromo-7-nitro-9H-fluoren-9-one (3) (9.0 g, 0.03 mol),
(M+H ): m/z = 323.2.
Pd (dba) (0.6 g, 0.655 mmol), BINAP (1.3 g, 2.087 mmol), t-
2
3
BuONa (3.3 g, 0.0343 mol), and 1,4-diazobicylco[3.2.2]nonane
3.0 g, 0.024 mol) were added to anhydrous 1,4-dioxane (150
4.2.6. 9-Oxo-9H-fluorene-1-carboxylic acid (8) [43]
(
A solution of CrO (138.0 g, 1.38 mol) in acetic acid (80 mL)
3
mL) sequentially and the reaction mixture was stirred overnight
at 80-85°C under an atmosphere of nitrogen. After cooling to
room temperature, the mixture was diluted with ethyl acetate
and water (120 mL) was added dropwise to a solution of
fluoranthene (40.0 g, 0.2 mol) in acetic acid (500 mL) at 85°C.
The solution was heated to reflux for 2 h and then poured into
water (3 L). The solid precipitate was filtered and dissolved in 2
M NaOH solution (0.6 L). Conc. HCl was added, and the yellow
solid obtained was filtered and dried in an oven at 100°C to
(150 mL) and filtered through Celite. The filtrate was adjusted to
pH = 8.0 with 10% K CO and then extracted with CH Cl . The
2
3
2
2
combined organic layers were dried over MgSO , filtered and
4
concentrated.
The
crude
product
2-(1,4-
provide pure 9-oxo-9H-fluorene-1-carboxylic acid (8) (29.5 g,
1
diazabicyclo[3.2.2]nonan-4-yl)-7-nitro-9H-fluoren-9-one (4) was
obtained as bluish violet solid (2.1 g, 20.4%) and used without
further purification. H NMR (400 MHz, CDCl ) δ 7.56 (d, J =
66.0%). H NMR (400 MHz, CDCl ) δ 8.20 (d, J = 7.84 Hz, 1H),
3
+
7.76-7.54 (m, 5H), 7.37 (t, J = 7.36 Hz, 1H); MS (M+H ): m/z =
1
225.10.
3
7
7
3
.24 Hz, 1H), 7.38 (d, J = 6.56 Hz, 1H), 7.34 (d, J = 2.8 Hz, 1H),
.14-7.09 (m, 2H), 6.78 (d, J = 8.16 Hz, 1H), 4.13-4.11 (m, 1H),
4.2.7. 7-Bromo-9-oxo-9H-fluorene-1-carboxylic acid (9)
The same method as described above for preparing 2 was used,
and 7-bromo-9-oxo-9H-fluorene-1-carboxylic acid (9) was
.67-3.65 (m, 1H), 3.61-3.58 (m, 1H), 3.15-3.14 (m, 4H), 3.04
+
(m, 2H), 2.14 (m, 2H), 1.78 (m, 2H); MS (M+H ): m/z = 350.16.
1
obtained as a yellow solid (19.5 g, 96.5%). H NMR (400 MHz,
4
.2.4.
2-Amino-7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-9H-
CDCl ) δ 8.22 (d, J = 7.04 Hz, 1H), 7.85 (s, 1H), 7.73-7.66 (m,
3
fluoren-9-one (5) [42]
3H), 7.43 (d, J = 7.84 Hz, 1H).
A mixture of 4 (2.1 g, 6.02 mmol), iron powder (1.7 g, 0.03
mol) in ethanol (80 mL), H O (54 mL) and conc. HCl (1.8 mL)
was heated to 80°C for 5 h. After complete conversion was
detected by TLC, the crude reaction mixture was adjusted to pH
4.2.8. Tert-butyl 2-bromo-9-oxo-9H-fluoren-8-ylcarbamate (10)
A solution of 7-bromo-9-oxo-9H-fluorene-1-carboxylic acid
(9) (6 g, 19.87 mmol) in t-BuOH (10 mL) and toluene (60 mL)
2
=
8.0 with a 10% solution of K CO , diluted with ethyl acetate
was treated with Et N (4.1 mL, 29.8 mmol) and DPPA (6.4 mL,
2
3
3
(
150 mL) and filtered through Celite. The extract was dried and
29.8 mmol). The reaction mixture was refluxed for 24 h and then
cooled to RT. The solvent was removed under vacuum, and the
crude product was purified by flash column chromatography
(PE:EA = 30:1) to provide the title compound tert-butyl 2-
concentrated under vacuum. The obtained solid was dissolved in
dichloromethane and washed with water. The organic layer was
dried over Na SO and filtered, and the solvent was removed
2
4
1
under vacuum to provide the title product 2-amino-7-(1,4-
bromo-9-oxo-9H-fluoren-8-ylcarbamate (10) (5.5 g, 74%). H
diazabicyclo[3.2.2]nonan-4-yl)-9H-fluoren-9-one (5) (1.5 g,
NMR (400 MHz, CDCl ) δ 9.25 (s, 1H), 8.15 (d, J = 8.56 Hz,
3
1
8
7
2
7
0%). H NMR (400 MHz, CDCl ) δ 7.12 (d, J = 8.24 Hz, 1H),
1H), 7.72 (d, J = 1.56 Hz, 1H), 7.60 (dd, J = 7.92 Hz, 1.72 Hz,
1H), 7.43 (t, J = 7.56 Hz, 1H), 7.37 (d, J = 7.84 Hz, 1H), 7.10 (d,
J = 7.24 Hz, 1H), 1.55 (s, 9H).
3
.08 (d, J = 7.92 Hz, 1H), 7.01 (d, J = 2.44 Hz, 1H), 6.88 (d, J =
.2 Hz, 1H), 6.70 (dd, J = 8.24 Hz, 2.52 Hz, 1H), 6.64 (dd, J =
.88 Hz, 2.24 Hz, 1H), 4.01 (s, 2H), 3.51-3.48 (m, 2H), 3.13-3.06
(
m, 4H), 3.01-2.94 (m, 2H), 2.12-2.05 (m, 2H), 1.75-1.67 (m,
4.2.9. 1-Amino-7-bromo-9H-fluoren-9-one (11)
HCl (1 M, 22 mL, 21.6 mmol) was added to a stirred solution
of tert-butyl 2-bromo-9-oxo-9H-fluoren-8-ylcarbamate (10) (808
13
2
1
1
3
H); C NMR (101 MHz, CDCl ) δ 194.18, 148.14, 144.99,
3
35.02, 134.79, 134.56, 132.85, 119.00, 118.94, 117.11, 110.32,
+
08.94, 55.59, 50.91, 45.26, 43.25, 25.50; MS (M+H ): m/z =
mg, 2.16 mmol) in CH CN (80 mL) and heated to 80°C for 4 h.
3
20.2.
The reaction mixture was cooled to RT, adjusted to pH = 12 with
1
M NaOH and extracted with ethyl acetate. The combined
4
.2.5.
2-(1,4-Diaza-bicyclo[3.2.2]nonan-4-yl)-7-fluoro-9H-
organic layers were dried over Na SO , filtered and concentrated
2
4
fluoren-9-one (6)
A mixture of 5 (1.9 g, 6 mmol) in HBF (30 mL) was cooled to
under vacuum. 1-Amino-7-bromo-9H-fluoren-9-one (11) was
obtained as a yellow solid (500 mg, 84.5%). H NMR (400 MHz,
1
4
0
-5°C and stirred for 5 min. A cold solution of NaNO (0.5 g, 7.2
CDCl ) δ 7.71 (d, J = 1.4 Hz, 1H), 7.55 (dd, J = 7.8 Hz, 1H), 7.35
2
3
mmol) in H O (15 mL) was added dropwise to the above solution
(d, J = 7.9 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 7.1 Hz,
1H), 6.52 (d, J = 8.4 Hz, 1H), 5.54 (s, 2H); MS (M+H ): m/z =
273.99, 275.98.
2
+
and stirred for 30 min. The precipitate was filtered and washed
with ethanol and tert-butyl methyl ether. The obtained diazonium
tetrafluoroborate was dried and refluxed at 120°C in xylene.
After complete convention was detected, the reaction system was
4
.2.10. 7-bromo-1-nitro-9H-fluoren-9-one (12)
brought to RT and dissolved in CH Cl . The organic layer was
2
2
A solution of trifluoroacetic anhydride (2.736 mL, 19.68
dried over Na SO , filtered and concentrated under vacuum. The
2
4
mmol) in CH Cl (2 mL) was added dropwise to 30% H O
2
2
2
2
crude product was purified by flash column chromatography
CH Cl :CH OH = 25:1) to provide the final compound 2-(1,4-
(16.95 mmol, 16.95 mmol) at 0°C and stirred for 1.5 h. 1-Amino-
(
2
2
3
7-bromo-9H-fluoren-9-one (11) (500 mg, 1.824 mmol) in CH Cl
2
2
diaza-bicyclo[3.2.2]nonan-4-yl)-7-fluoro-9H-fluoren-9-one (6)
(4 mL) was added dropwise to the above mixture and warmed to
1
(
1.2 g, 67.3%) as a purple solid. H NMR (400 MHz, CDCl ) δ
3
RT. After 3-9 was consumed completely, the mixture was
8

quenched by adding water. The phases separated, and the
solution of 2-nitropyridin-3-ol (16) (5 g, 35.7 mmol) in CH OH
(50 mL) and the mixture was stirred at RT for 30 min. After the
3
ACCEPTED MANUSCRIPT
aqueous layer was extracted three times with CH Cl . The
2
2
combined organic layers were dried over Na SO , filtered and
reaction system cooling to 0°C, Br (6.3 g, 39.4 mmol) was added
2
4
2
concentrated under vacuum. The crude product was purified by
flash column chromatography (PE:EA = 10:1) to provide the title
dropwise with stirring. After 16 was consumed, the mixture was
quenched by adding a solution of 10% Na SO and then cooled
2
3
compound 7-bromo-1-nitro-9H-fluoren-9-one (12) (212 mg,
to -15°C. Yellow solid was precipitated, filtered and washed with
1
3
7
7
8.2%). H NMR (400 MHz, CDCl ) δ 7.85 (d, J = 1.6 Hz, 1H),
cold CH OH. The title compound was obtained as a yellow solid
3
3
1
.78-7.76 (m, 1H), 7.72-7.70 (m, 1H), 7.68-7.66 (m, 1H), 7.64-
(4.2 g, 53%). H NMR (400 MHz, DMSO) δ 7.85 (d, J = 8.64 Hz,
+
+
.62 (m, 1H), 7.49 (d, J = 7.9 Hz, 1H). MS (M ): m/z = 303, 305.
1H), 7.66(d, J = 8.64 Hz, 1H); MS (M+H ): m/z = 216.97.
4
.2.11.
7-(1,4-Diaza-bicyclo[3.2.2]nonan-4-yl)-1-nitro-9H-
4.2.15. 6-bromo-2-nitropyridin-3-ol (18)
fluoren-9-one (13)
Under an atmosphere of nitrogen, zinc powder (1.5 g, 22.9
Under an atmosphere of nitrogen, Pd (dba) (0.6 g, 0.655
mmol) and NH Cl (1.5 g, 22.9 mmol) were added to the solution
2
3
4
mmol) and BINAP (61 mg, 0.099 mmol) were stirred in
anhydrous toluene (2 mL) for 15 min at 90°C. The solution was
cooled to RT, and a mixture of 7-bromo-1-nitro-9H-fluoren-9-
one (12) (100 mg, 0.329 mmol), Cs CO (426 mg, 1.31 mmol),
of 2-nitropyridin-3-ol (17) (1 g, 4.6 mmol) in C H OH (20 mL).
2
5
The reaction was heated to 50°C and stirred for 16 h. The crude
reaction mixture was filtered and purified by flash column
chromatography (PE:EA = 5:1) to provide the title compound 6-
2
3
1
and 1,4-diazobicylco[3.2.2]nonane (125 mg, 0.989 mmol) in
anhydrous toluene (2 mL) was added. The reaction mixture was
heated to 80-85°C and stirred overnight. After cooling to room
temperature, the mixture was quenched by adding water. The
phases separated and the aqueous layer extracted three times with
CH Cl . The combined organic layers were dried over Na SO ,
bromo-2-nitropyridin-3-ol (18) (524 mg, 60%). H NMR(400
MHz, DMSO) δ 9.70(s, 1H), 6.74(d, J = 7.8 Hz, 1H), 6.49 (d, J =
+
7.8 Hz, 1H), 5.91(s, 1H); MS (M+H ): m/z = 188.99.
4.2.16. 5-bromooxazolo[4,5-b]pyridine-2-thiol (20)
KOH (191 mg, 4.76 mmol) was added to a stirred mixture of
2
2
2
4
6
-bromo-2-nitropyridin-3-ol (18) (0.3 g, 1.59 mmol) in C H OH
filtered and concentrated under vacuum
purified by flash column chromatography (CH Cl :CH OH =
.
The crude product was
2 5
(10 mL) under an argon atmosphere. Carbon disulfide (2.42 g,
2
2
3
3
1.7 mmol) was added and the reaction mixture was heated to
2
0:1) to provide the title compound 7-(1,4-diaza-
reflux for 16 h. The reaction mixture was evaporated to dryness,
and water (250 mL) was added followed by HCl until the pH was
~
bicyclo[3.2.2]nonan-4-yl)-1-nitro-9H-fluoren-9-one (13) (48 mg,
1
4
1.8%). H NMR (400 MHz, CDCl ) δ 7.52-7.49 (m, 2H), 7.36
3
3. The formed precipitate was separated by filtration, washed
(d, J = 7.9 Hz, 2H), 7.10 (s, 1H), 6.82 (t, J = 7.6 Hz, 1H), 4.13 (s,
with H O, and dried to provide the crude product 19 (0.26 g).
1
1
H), 3.63-3.62 (m, 2H), 3.18-3.04 (m, 6H), 2.31 (s, 2H), 1.83-
.82 (m, 2H); MS (M+H ): m/z = 350.37.
2
+
K CO was added to a stirred mixture of the above product
2
3
(0.26 g, 1.13 mmol) in DMF (10 mL) under argon. Then methyl
iodide (1.24 mmol) was added dropwise at 0°C. The mixture was
4
.2.12.
7-(1,4-Diaza-bicyclo[3.2.2]nonan-4-yl)-1-amino-9H-
stirred at RT for 2 h and evaporated. H O (60 mL) and ethyl
fluoren-9-one (14)
The same method as described above for preparing 5 was
used, and 7-(1,4-diaza-bicyclo[3.2.2]nonan-4-yl)-1-amino-9H-
2
acetate (20 mL) were added to the residue, and the mixture was
stirred for 5 min. The organic layer was separated, and the
aqueous layer was extracted with ethyl acetate (3 × 200 mL). The
fluoren-9-one (14) was obtained as a purple solid (1.5 g, 80%).
1
combined organic layers were dried over Na SO and evaporated
H NMR (400 MHz, CDCl ) δ 7.30 (d, J = 8.28 Hz, 1H), 7.16-
2
4
3
to provide the title compound 5-bromooxazolo[4,5-b]pyridine-2-
7
2
1
3
.12 (m, 1H), 7.06 (d, J = 2.48 Hz, 1H), 6.75 (dd, J = 8.28 Hz,
.52 Hz, 1H), 6.67 (d, J = 7.04 Hz, 1H), 6.35 (d, J = 8.28 Hz,
H), 5.43 (s, 2H), 4.10 (s, 1H), 3.58 (t, J = 5.48 Hz, 2H), 3.19-
.13 (m, 3H), 3.07-3.00 (m, 2H), 2.14-2.11 (m, 2H), 1.80-1.74
1
thiol (20) (180 mg, 65%). H NMR(400 MHz, DMSO) δ 8.06 (d,
J = 8.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 2.79 (s, 3H); MS
+
(
M+H ): m/z = 246.93.
13
(m, 3H); C NMR (100 MHz, CDCl ) δ 195.66 (s), 150.20 (s),
3
4
.2.17. 5-bromo-2-(methylthio)oxazolo[4,5-b]pyridine (21)
,4-Diazabicyclo[3.2.2]nonane (200 mg, 1.58 mmol) was
added to a solution of 5-bromooxazolo[4,5-b]pyridine-2-thiol (20)
1
1
4
47.18 (s), 137.02-136.73 (m), 136.37 (s), 131.22 (s), 121.42 (s),
16.55 (s), 115.39 (s), 108.58 (s), 108.48 (s), 57.06 (s), 51.87 (s),
6.56 (s), 44.64 (s), 26.91 (s); MS (M+H ): m/z = 320.16.
1
+
(
466 mg, 1.9 mmol) and NEt (321 mg, 3.17 mmol) in i-PrOH
3
(
10 mL). The mixture was placed in an oil bath at 100°C and the
4
.2.13.
7-(1,4-Diaza-bicyclo[3.2.2]nonan-4-yl)-1-fluoro-9H-
solvent was evaporated. The mixture was allowed to stir neat at
0°C overnight. Upon cooling to RT, the mixture was purified by
flash column chromatography (EA) to provide the title compound
fluoren-9-one (15)
9
The same reaction as described above for preparing 6 was used,
and 7-(1,4-diaza-bicyclo[3.2.2]nonan-4-yl)-1-fluoro-9H-fluoren-
9
NMR (400 MHz, CDCl ) δ 7.39-7.34 (m, 1H), 7.32 (d, J = 8.36
Hz, 1H), 7.11 (d, J = 7.36 Hz, 1H), 7.09 (d, J = 2.48 Hz, 1H),
6
3
1
5
4
7
2
2
-bromo-2-(methylthio)oxazolo[4,5-b]pyridine (21) (250 mg,
-one (15) was obtained as a purple solid (446 mg, 34.0%). H
1
8.8%). H NMR (400 MHz, DMSO) δ 7.68 (d, J = 8.12 Hz, 1H),
3
.15 (d, J = 8.12 Hz, 1H), 4.43-4.42 (m, 1H), 3.90 (t, J = 5.56 Hz,
H), 3.09 (t, J = 5.76 Hz, 2H), 3.04-2.95 (m, 4H), 2.10-2.07 (m,
.80-6.74 (m, 2H), 4.10 (s, 1H), 3.60 (t, J = 5.76 Hz, 2H), 3.19-
.12 (m, 4H), 3.05-2.99 (m, 2H), 2.15-2.10 (m, 2H), 1.82-1.73
+
H), 1.84-1.76 (m, 2H); MS (M+H ): m/z = 323.905.
13
(m, 2H); C NMR (101 MHz, CDCl ) δ 191.17 (s), 158.08 (s),
3
4
3
.2.18. 2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-(2-fluoropyridin-
-yl)oxazolo[4,5-b]pyridine (24)
1
50.54 (s), 137.22 (s), 137.14 (s), 135.82 (s), 130.97 (s), 121.76
(s), 117.27 (s), 115.33 (s), 115.12 (s), 114.98 (s), 109.26 (s),
19
Under an atmosphere of nitrogen, Cs CO (303 mg, 0.93 mmol)
5
6.85 (s), 51.62 (s), 46.36 (s), 44.39 (s), 29.70 (s), 26.65 (s);
F
2
3
+
and Pd(dppf)Cl (45 mg, 0.062 mmol) were added to a solution of
NMR (376 MHz, CDCl ) δ -114.11 (s); MS (M+H ): m/z =
3
2
3
5
0
2
-bromo-2-(methylthio)oxazolo[4,5-b]pyridine (21) (100 mg,
.31 mmol), 2-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
-yl)pyridine (22) (70 mg, 0.314 mmol) in 1,4-dioxane and
23.2.
4
.2.14. 2-nitropyridin-3-ol (17)
Under an atmosphere of nitrogen, a solution of CH ONa (2.89
heated to 85-90°C. After complete convention was detected, the
residue was purified by flash column chromatography (PE:EA =
3
g, 53.5 mmol) in CH OH (15 mL) was added dropwise to a
3
9

1
0:1)
to
provide
the
title
compound
2-(1,4-
ɑ7-nAChR assay: the saturation binding assay was conducted
using the membranes (1.5 mg) prepared above incubated in Tris-
ACCEPTED MANUSCRIPT
diazabicyclo[3.2.2]nonan-4-yl)-5-(2-fluoropyridin-3-
yl)oxazolo[4,5-b]pyridine (24) as yellow solid (89.4 mg, 85%).
HCl buffer at 37°C for 150 min with different concentrations of
[ I]α-bungarotoxin (10 µL) in a final volume of 500 µL.
1
125
H NMR(400 MHz, CDCl ) δ 8.72 (td, J = 8.82 Hz, 2 Hz, 1H),
3
8
.18 (dt, J = 1.76 Hz, 4.32 Hz, 1H), 7.61 (dd, J = 8.24 Hz, 1.32
Nonspecific binding was estimated by adding α-bungarotoxin (2
µM, 100 µL). The binding was terminated by rapid vacuum
filtration through glass fiber filters (Whatman GF/B, presoaked
in 0.5% (v/v) polyethyleneimine for 150 min) using an Mp-48T
cell harvester (Brandel, Gaithersburg, MD), and the filters were
washed with ice-cold Tris-HCl buffer 3 times. The radioactivity
retained on the filters was counted using an automatic γ-counter.
The reference ligand MLA and the synthesized compounds were
Hz,1H), 7.49 (d, J = 8.2 Hz, 1H), 7.30 (td, J = 5.94 Hz, 2.04 Hz,
1
2
CDCl ) δ163.54, 161.83, 159.45, 158.78, 146.72, 146.02, 141.72,
1
4
H), 4.61 (s, 1H), 3.99(m, 2H), 3.21-3.13 (m, 4H), 3.06-2.99(m,
13
H), 2.18-2.16 (m, 2H), 1.88-1.80 (m, 2H); C NMR (100 MHz,
3
41.05, 122.63, 122.37, 122.01, 116.37, 114.92, 56.96, 50.73,
+
6.32, 44.33, 26.82; MS (M+H ): m/z = 340.15.
125
4
3
.2.19. 2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-(6-fluoropyridin-
-yl)oxazolo[4,5-b]pyridine (25)
The same method as described above for preparing 24 was
evaluated in the presence of [ I]α-bungarotoxin (0.4 nM). These
assays were performed independently in triplicate. Binding assay
results were analyzed using GraphPad Prism 5.0 software, and
the Ki values were calculated using the Cheng-Prusoff equation.
α4β2-nAChRs assay: the affinity of the compounds for α4β2-
nAChRs was carried out using a modification method described
used,
and
2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-(6-
fluoropyridin-3-yl)oxazolo[4,5-b]pyridine (25) was obtained as
yellow solid (53 mg, 50%). H NMR (400 MHz, CDCl ) δ 8.80
1
3
3
(
=
2
d, J = 2.32 Hz, 1H), 8.52 (td, J = 7.8 Hz, 2.52 Hz, 1H), 7.50 (d, J
8.16 Hz, 1H), 7.35 (d, J = 8.12 Hz, 1H), 6.99 (dd, J = 8.52 Hz,
.8 Hz, 1H), 4.65 (s, 1H), 4.03 (t, J = 5.36 Hz, 2H), 3.25-3.19
above. In brief, [ H]cytisine (1nM) as the radioligand competed
with test compounds. Nonspecific binding was measured by
adding 1µM cytisine. Incubation and filtration were performed as
described above. The samples were counted in a liquid
13
(m,4H), 3.10-3.04 (m, 2H), 2.22 (m, 2H), 1.93-1.86 (m, 2H);
C
NMR (100 MHz, CDCl ) δ 164.88, 163.48, 162.49, 158.88,
scintillation counter and the data was analyzed using the method
mentioned above.
3
1
1
3
49.03, 145.90, 141.07, 139.96, 133.43, 115.37, 112.24, 109.51,
09.14, 55.83, 50.57, 46.30, 26.43, 8.70; MS(M+H ): m/z =
40.16.
+
4.5. Patch clamp electrophysiology
4
.3. Radiolabeling
Agonistic effects were assessed by a manual patch clamp
technique performed commercially by Beijing Ice Bioscience
NIC.
18
[
F]fluoride trapped on a QMA cartridge was transferred into
a high-pressure digestion tank with a solution of Kryptofix
22/K C O (a mixture of 15 mg Kryptofix 222 in 0.7 mL
2
4.6. hERG assay
2
2
4
CH CN and 2 mg K C O in 0.3 mL H O). The solvent was
3
2
2
4
2
removed at 100°C under a stream of nitrogen gas. The residue
The hERG inhibitory activity of the four derivatives was
measured by a traditional patch clamp technique performed
commercially by Beijing Ice Bioscience NIC. In general,
HEK293 cells stably expressing hERG potassium channels were
routinely grown at 37°C under a 5% CO2 atmosphere in
Dulbecco’s modified eagle’s medium/nutrient mixture (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum and
was azeotropically dried with 0.5 mL of anhydrous CH CN three
3
times at 100°C under a stream of nitrogen gas. Nitro precursor 13
(
2 mg) in anhydrous DMSO (0.3 mL) was added to the above
mentioned high-pressure digestion tank and heated to 160°C for
0 min. The mixture was diluted with water (2×10 mL) and
3
passed through a Sep-Pak C18 cartridge. After washing the
column with additional water (10 mL), the crude product was
TM
0.2 mg/mL geneticin. After culture, TrypLE Express solution
3
eluted with CH CN (2 mL), and the solvent was removed under
was used to separate the cells, then 3×10 cells were spread onto
3
vacuum. The residue was dissolved in CH CN and purified by
a cover glass, and the cell electrophysiological activity was
detected after incubation for 18 h in a 24 well plate. The ligands
were evaluated until the hERG channel currents were stable. All
of the experiments were performed independently 3 times.
3
HPLC (mobile phase: CH CN/H O (0.2% NH OAC) = 28/72,
3
2
4
flow rate = 4 mL/min, λ = 280 nm). Identification was achieved
by analytical HPLC (mobile phase: CH CN/H O (0.2%
3
2
NH OAC) = 28/72, flow rate = 1 mL/min, λ = 280 nm). The total
4
synthesis time was approximately 60 min. The radiochemical
yield was 13.1% (decay not corrected), and the radiochemical
purity was greater than 98%.
4.7. Acute toxicity assay
Kunming mice (half male and half female, 18-20 g, n = 60)
were fed for 3 days, and after 12 h of fasting (food but not water
was withheld), the mice were weighed and randomly divided into
4
.4. In vitro binding assay
6
groups. Every mouse was injected with different concentrations
Preparation of rat membranes: SD rats (female, 180 - 200 g)
of 15 (0.1 mL) via the tail vein, and the control groups were
injected only with the vehicle solution. The conditions of the
mice, including behavioral activities, food intake, changes in
body weight, and signs of toxicity or death, were observed over a
span of 14 days. The LD50 value was obtained by the weighted
probit analysis developed by Bliss.
were used for the experiment. After the rats were sacrificed by
decapitation, the brains were rapidly removed from the skulls and
dissected on ice. The cerebral cortexes (rich in ɑ7 nAChRs) were
collected, homogenized at 4°C in 15 volumes of ice-cold Tris-
HCl buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM
CaCl , 1 mM MgCl , pH = 7.4) and centrifuged at 48000 g for 20
2
2
min at 4°C. The supernatant was discarded, and the pellets were
resuspended, homogenized and centrifuged as above three times.
Subsequently, the pellets were resuspended into 10 volumes of
buffer and kept at -80°C. Protein concentrations were measured
using the method of Lowry et al. [44]
4.8. Partition coefficient determination
The partition coefficient of [ F]15 was determined by
18
measuring the distribution of the radioligand in n-octanol and
18
phosphate buffered saline (PBS). [ F]15 (10 µCi) dissolved in
0.1 mL of saline was added to a centrifuge tube, to which 0.8 mL
of PBS saturated with n-octanol and 0.9 mL n-octanol saturated
Ligand-binding assay:
1
0

with PBS were added. The tube was vortexed for 1.0 min,
position. Images were acquired at 15, 30 and 60 min post-
injection.
ACCEPTED MANUSCRIPT
followed by centrifugation for 5 min at 7000 rpm. Then, two
samples (0.1 mL each) from each phase were counted in an
automatic γ-counter. The partition coefficient was expressed as
log P which was calculated using the following equation: log P =
log(counts (n-octanol)/counts (PBS)). Samples (0.45 mL) from
the n-octanol layer were repartitioned until consistent distribution
coefficient values were obtained. The measurement was repeated
three times in triplicate.
Compliance with ethical standards
All protocols requiring the use of mice were approved by the
Animal Care Committee of Beijing Normal University.
Author contributions
⊥
4
.9. In vitro stability studies
Shuxia Wang and Yu Fang contributed equally to this work.
18
In vitro stability in fetal bovine serum and saline: [ F]15 (10
µCi) was incubated in fetal bovine serum (100 µL) and saline
100 µL) at 37°C and RT, respectively. The incubation mixtures
Conflicts of interest
(
The authors declare no conflict of interest.
were sampled at 60 min and 120 min and loaded onto the HPLC
system for analysis. Specifically, the fetal bovine serum sample
was treated using the following procedure: after incubating, the
serum protein was precipitated by adding acetonitrile (200 µL),
and the sample was centrifuged for 5 min at 7000 rpm. The
supernatant was filtered through an organic Millipore filter (0.22
µm) for HPLC analysis.
Acknowledgments
The work was financially supported by the National Major
Scientific and Technological Special Project for “Significant
New Drugs Development” (Grant Nos. 2014ZX09507007-001
and 2014ZX09507007-003), the National Science and
Technology Support Program (Grant No. 2014BAA03B03) and
the National Natural Science Foundation of China (Grant Nos.
21371026). We also thank the Nuclear Medicine Department of
Peking Cancer Hospital (Beijing, China) for providing the
fluoride-18 nuclide and allowing us to use the micro-PET/CT
scanner.
4
.10. Biodistribution in tissues and organs in mice
Normal Kunming mice (female, 18-22 g, n = 5) were injected
18
with [ F]15 (10 µCi, in saline (0.1 mL) containing 5% DMSO)
via the tail vein and sacrificed by decapitation at 5, 15, 30, 60 and
9
0 min post-injection. The tissue and organs of interest were
removed, weighed and counted by an automatic γ-counter. The
percent dose per gram of wet tissue (%ID/g) was calculated by
comparison of the tissue counts with suitably diluted aliquots of
the injected material. The values were expressed as the mean ±
SD (n = 5).
Appendix A. Supplementary data
Data analysis of the patch clamp electrophysiological assay,
1
13
and acute toxicity assay, and H NMR, C NMR and MS spectra
are listed in the supporting information.
4
.11. Biodistribution in the brain regions of mice
References
Normal Kunming mice (female, 28-32 g, n = 5) were injected
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0 and 90 min post-injection. The brains were rapidly removed
from the skulls and then dissected on ice into the cortex,
hippocampus, striatum, superior and inferior colliculus, thalamus,
cerebellum and rest of the brain. The brain regions were weighed
and the radioactivity was determined with an automatic γ-
counter. The data was analyzed as described as above.
18
[
[
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ACCEPTED MANUSCRIPT
1
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1
3

ACCEPTED MANUSCRIPT
1
A class of 1,4-diazobicylco[3.2.2]nonane derivatives was synthesized and evaluated
as α7-nAChR ligands.
2
3
Five ligands displayed high binding affinity (K = 0.001-25 nM) for α7-nAChRs.
i
[ F]15 displayed high binding affinity, selectivity for α7-nAChRs and good
18
pharmacokinetics in mice.